非洲猪瘟病毒无标签重组B602L抗原制备与鉴定

Preparation and identification of the tag-free B602L protein for detection of African swine fever virus antibody

为了获得无标签重组抗原用于非洲猪瘟病毒(ASFV)抗体检测,本研究将B602L与类弹性蛋白多肽(ELP)基因进行融合表达,利用简单、经济的相变循环(ITC)纯化ELP-B602L融合蛋白,用烟草蚀纹病毒(TEV)蛋白酶活性包涵体切除ELP标签,再用ITC回收重组B602L蛋白;用抗体阳性猪血清对重组B602L进行鉴定;以重组B602L蛋白为包被抗原进行ELISA鉴定。结果显示重组大肠杆菌能正确表达ELP-B602L融合蛋白,纯化融合蛋白纯度大于85%;TEV蛋白酶活性包涵体切除ELP标签的效率大于90%,回收的重组B602L蛋白纯度大于90%,能与抗体阳性猪血清反应;以重组B602L蛋白为包被抗原建立的ELISA与抗体阳性血清反应为阳性,与抗体阴性血清反应为阴性,OD450值与血清稀释倍数具有线性相关性。

To obtain a tag-free recombinant antigen for detection of African swine fever virus(ASFV)antibodies,the B602L gene was fused with elastin-like polypeptide(ELP)and expressed on E.coli as an ELP-B602L fusion protein.The fusion protein was purified by simple,economical inverse transition(ITC)and the ELP tag was cleaved with the active inclusion body of tobacco etch virus(TEV)protease.The ELP tag was removed by centrifugation and the tag-free B602L protein was recovered by ITC.The results showed that the ELP-B602L fusion protein was expressed correctly on E.coli and purified to more than 85%purity.The fusion protein was cleaved by TEV protease at a 90%cleavage efficiency.After removing the ELP tag,the recovered recombinant B602L protein was of more than 90%purity which could be identified by B602L-specific antiserum.The recombinant B602L protein reacted negatively with the antibody-negative serum and positively with the antibody-positive serum in ELISA,with a good linear correlation between the OD450 value and serum dilution.These data suggested that the tag-free B602L antigen might be used to detect ASFV antibodies by ELISA and Western blotting.